The HIV-1 accessory proteins Vpu antagonize a variety of host restriction factors, thus leading to the escape of host immune surveillance and the promotion of virus release. However, the interaction between Vpu and the host proteins has not been fully clarified. Here, we utilized APEX2-based proximity labeling and transmission electron microscope, as well as Immunoprecipitation-Mass spectrometry to characterize the interaction between HIV-1 Vpu and host proteins. We identified 21 cellular targets of Vpu and found the targets were enriched in RNA transport and spliceosome pathway.